CN114720568A - Method for establishing fingerprint spectrum of radix pseudostellariae medicinal material and application thereof - Google Patents
Method for establishing fingerprint spectrum of radix pseudostellariae medicinal material and application thereof Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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Abstract
The invention provides a method for establishing a fingerprint of a radix pseudostellariae medicinal material and application thereof, wherein the method adopts a high performance liquid chromatography, and the conditions of the high performance liquid chromatography comprise: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is used as a mobile phase A, water is used as a mobile phase B to form a mixed mobile phase, and the volume ratio of the mobile phase A to the mobile phase B in the mixed mobile phase within 0-55 min is from 5: 95 to 90: elution is carried out with a gradient of 10; the detection wavelength was 203 nm. On the other hand, the invention also provides a method for detecting the active ingredients of the radix pseudostellariae medicinal material. The invention also provides an HPLC standard fingerprint of the radix pseudostellariae medicinal material. The invention establishes a determination method for simultaneously determining multiple components in the radix pseudostellariae, the sample pretreatment is simple, the chromatographic conditions are specific to multiple types of compounds, and the identification of 8 compounds is completed.
Description
Technical Field
The invention relates to the field of pharmaceutical analysis, in particular to a fingerprint spectrum establishing method of a radix pseudostellariae medicinal material, and also relates to a standard fingerprint spectrum obtained by the fingerprint spectrum establishing method and application of the standard fingerprint spectrum in the aspects of active ingredient detection and quality control of the radix pseudostellariae medicinal material.
Background
The traditional Chinese medicine fingerprint refers to a chromatogram or a spectrogram of a certain kind or several kinds of characteristic components shared by a certain traditional Chinese medicine or raw material medicines and preparations thereof. Under the condition that most of the effective components of the traditional Chinese medicine are not very clear at the present stage, the establishment of the fingerprint has important significance for effectively controlling the quality of the traditional Chinese medicine and the quality of the raw material medicines and preparations thereof.
Radix Pseudostellariae is dry root tuber of Pseudostellaria heterophylla (Miq.) Pax ex Pax et Hoffm of Caryophyllaceae, and is distributed in Liaoning, inner Mongolia, Hebei, Shanxi, Shandong, Jiangsu, Anhui, Zhejiang, Jiangxi, Henan, Hubei, Hunan, Sichuan and other places. It has effects of invigorating qi and spleen, promoting fluid production and moistening lung, and can be used for treating spleen deficiency, asthenia, anorexia, asthenia after illness, deficiency of both qi and yin, spontaneous perspiration, thirst, and dry cough due to lung dryness.
At present, the research on the fingerprint of the radix pseudostellariae medicinal material has been reported. For example, in the patent application "a preparation method of radix pseudostellariae extract and a radix pseudostellariae extract prepared by the preparation method and a detection method" (201811382057.7), the following quality control method of the radix pseudostellariae extract is established: a. a method for measuring the UV content of the radix pseudostellariae polysaccharide based on a phenol-sulfuric acid method; b. a saponin UV content determination method based on a vanillin glacial acetic acid perchloric acid method; c. a method for measuring content of cyclic peptide of radix Pseudostellariae based on HPLC is provided. The patent application 'a method for constructing UPLC characteristic spectrum of pseudostellaria root medicinal material and pseudostellaria root standard decoction and an identification method' (201910914035.9) establishes a method for detecting variable waves by adopting ethanol ultrasonic extraction, a C18 chromatographic column, an acetonitrile-phosphoric acid aqueous solution system and 200-180-240 nm-. However, no standard characteristic profile is disclosed in this patent application, no spectral peaks are identified, and no compound components are identified. The Chile Biaobiao et al reported in the research on HPLC fingerprint of radix Pseudostellariae that the method of sample pretreatment by ethyl acetate ultrasonic extraction, polyamide column chromatography, gradient ethanol elution, C18 chromatographic column, acetonitrile-water system gradient elution and 203nm wavelength detection identifies 35 common peaks, and selects 20 common peaks as quality control items. This method achieves a higher organic phase ratio in a faster time, but the method does not perform compound peak identification. Liaofanping et al reported in the research on HPLC fingerprint of Pseudostellaria heterophylla Pacifica Linn that the analysis method of degreasing with ether, refluxing and extracting with methanol, chromatography with macroporous adsorption resin, elution with 90% ethanol for sample pretreatment, C18 chromatographic column, gradient elution with acetonitrile-water system and 203nm detection wavelength. The method is suitable for separating and detecting components with medium polarity to small polarity.
Obviously, the research only provides characteristic fingerprint peaks of some or a plurality of specific components, has certain defects in the recognition of basic substances of the radix pseudostellariae medicinal material, and has the defects of complex pretreatment of a sample, so that the application range of the sample is limited, and the process and the quality cannot be well controlled in the production process of the radix pseudostellariae raw material medicine and the preparation.
Therefore, based on the defects of the prior art, a fingerprint detection method of the radix pseudostellariae medicinal material capable of better controlling the quality is required to be established so as to solve the problem that the radix pseudostellariae medicinal material has no prospective method for comprehensively detecting the effective components.
Disclosure of Invention
Therefore, the invention aims to provide a method for establishing a fingerprint spectrum capable of comprehensively representing the quality of the radix pseudostellariae medicinal material and identifying compound peaks in the fingerprint spectrum, aiming at the defects that the identification rate of characteristic fingerprint peaks in the fingerprint spectrum of the radix pseudostellariae medicinal material is low and the process and quality control of the radix pseudostellariae medicinal material cannot be well realized at present, and the method can be used for comprehensively detecting the active ingredients of the radix pseudostellariae medicinal material. The invention also provides a standard fingerprint of the radix pseudostellariae medicinal material and identifies chemical components in the standard fingerprint, thereby realizing the monitoring of the whole process from the radix pseudostellariae medicinal material to the raw material medicinal material and preparation of the radix pseudostellariae medicinal material and the quality, facilitating the confirmation of the basic materials of the radix pseudostellariae medicinal material, the raw material medicinal material and the preparation of the radix pseudostellariae, and the evaluation of the production process and the quality, and providing a standard indication for the subsequent process improvement and perfection.
Aiming at the purposes, the technical scheme of the invention is as follows:
on one hand, the invention provides a method for establishing a fingerprint of a radix pseudostellariae medicinal material, which adopts a high performance liquid chromatography, wherein the conditions of the high performance liquid chromatography comprise the following steps: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is used as a mobile phase A, water is used as a mobile phase B to form a mixed mobile phase, and the volume ratio of the mobile phase A to the mobile phase B in the mixed mobile phase within 0-55 min is from 5: 95 to 90: elution is carried out with a gradient of 10; the detection wavelength was 203 nm.
According to the fingerprint establishment method of the present invention, preferably, the gradient is as follows:
according to the fingerprint spectrum establishing method, the flow rate of the mixed mobile phase is preferably 1 mL/min.
According to the fingerprint establishing method of the present invention, preferably, the conditions of the high performance liquid chromatography further include: the column temperature is 20-40 ℃, more preferably, the column temperature is 25 ℃; preferably, the sample size is 10. mu.L.
According to one embodiment of the invention, the fingerprint establishing method comprises the following steps:
(1) preparation of control solutions: taking radix Pseudostellariae cyclic peptide B reference substance, and adding methanol to obtain reference substance solution;
(2) preparation of a test solution: taking radix pseudostellariae medicinal material, adding methanol, carrying out ultrasonic treatment, filtering, and taking subsequent filtrate to obtain a test solution;
(3) the determination method comprises the following steps: and (3) respectively injecting the reference substance solution prepared in the step (1) and the test substance solution prepared in the step (2) into a high performance liquid chromatograph, and obtaining a high performance liquid chromatography spectrum according to the conditions of the high performance liquid chromatography.
Preferably, the fingerprint establishing method according to the present invention comprises the following steps:
(1) preparation of control solutions: taking a radix pseudostellariae cyclic peptide B reference substance, and adding methanol to prepare a reference substance solution containing 0.02mg of radix pseudostellariae cyclic peptide B per 1 mL;
(2) preparation of a test solution: taking 0.5g of radix pseudostellariae, adding 10mL of methanol, carrying out ultrasonic treatment for 10-60 minutes, such as 30 minutes, cooling, supplementing the lost weight with methanol, shaking up, filtering, and taking the subsequent filtrate to obtain a test solution;
(3) the determination method comprises the following steps: and (3) respectively injecting the reference substance solution prepared in the step (1) and the test substance solution prepared in the step (2) into a high performance liquid chromatograph, and obtaining a high performance liquid chromatography spectrum according to the conditions of the high performance liquid chromatography.
According to a specific embodiment of the present invention, the fingerprint establishing method comprises the following steps:
1. preparation of a reference solution: accurately weighing 1.00mg of radix Pseudostellariae cyclic peptide B reference substance in a 50mL measuring flask, adding methanol to constant volume to scale, and filtering (0.45 μm filter membrane);
2. preparing a test solution: pulverizing radix Pseudostellariae (sieving with 60 mesh sieve), weighing powder 0.5g, adding methanol 10mL, weighing, ultrasonic processing for 30min, taking out, supplementing with methanol, filtering (0.45 μm filter membrane), and collecting filtrate;
3. the determination method comprises the following steps: respectively and precisely taking 10 mu l of each of the reference solution prepared in the step (1) and the test solution prepared in the step (2), injecting into a high performance liquid chromatograph, and obtaining a high performance liquid chromatography spectrum according to the following conditions of the high performance liquid chromatography:
YMC-Triart C18 column (4.6 mm. times.250 mm, 5 μm, Japan YMC Co.); acetonitrile (A) -water (B) is used as a mixed mobile phase, and the elution gradient is shown in the table; the flow rate is 1 mL/min; the detection wavelength is 203 nm; the column temperature was 25 ℃.
Preferably, the fingerprint spectrum establishing method further comprises the steps of obtaining high performance liquid chromatography spectrums of a plurality of radix pseudostellariae medicinal material samples according to the conditions of the high performance liquid chromatography, checking and matching retention time of common chromatographic peaks in the high performance liquid chromatography spectrums by selecting reference peaks in the high performance liquid chromatography spectrums, calculating similarity, and generating common peak mode spectrums.
On the other hand, the invention also provides a method for detecting the active ingredients of the radix pseudostellariae medicinal material, which comprises the following steps: obtaining a high performance liquid chromatography fingerprint by adopting the radix pseudostellariae medicinal material fingerprint establishing method, obtaining a high performance liquid chromatography of a to-be-detected sample of the radix pseudostellariae medicinal material according to the conditions of the high performance liquid chromatography, and comparing the high performance liquid chromatography and the high performance liquid chromatography.
Preferably, the method for detecting the active ingredients of the radix pseudostellariae medicinal material further comprises the step of identifying the ingredients of the sample to be detected of the radix pseudostellariae medicinal material by adopting a mass spectrometry method.
The radix pseudostellariae medicinal material fingerprint provided by the invention is characterized in that the fingerprint is an HPLC standard fingerprint, and the standard fingerprint of the radix pseudostellariae medicinal material formed by common characteristic peaks of samples is obtained by analyzing and comparing a sample solution of the radix pseudostellariae medicinal material according to the high performance liquid chromatography. The fingerprint spectrum can be compared by introducing different batches of radix pseudostellariae medicinal material HPLC spectra into related software, such as a traditional Chinese medicine chromatogram fingerprint spectrum similarity evaluation system. The retention time of the common peak can be corrected and matched by selecting a reference peak, the similarity is calculated, and a 'median method' is adopted to generate the common mode atlas.
The radix pseudostellariae medicinal material fingerprint spectrum provided by the invention at least comprises 12 common characteristic peaks, wherein the No. 5 peak: pseudostellarin a; peak No. 6: pseudostellarin B; peak No. 7: pseudostellaria cyclic peptide B; peak No. 8: pseudostellarin C; peak No. 9: pseudostellarin D; peak No. 10: pseudostellaria cyclic peptide A; peak No. 11: pseudostellarin G; peak No. 12: pseudostellarine E. The relative retention time ranges of the 12 common characteristic peaks relative to the reference peak pseudostellaria cyclic peptide B peak are respectively as follows: 0.505, 0.675-0.678, 0.709, 0.739, 0.789-0.790, 0.941, 1.000, 1.067, 1.217-1.221, 1.956-1.957, 2.030, 2.146-2.147.
The percentage ranges of the peak areas of the 12 common characteristic peaks in the total peak area are respectively as follows: 1.01 to 3.70, 3.72 to 9.03, 4.37 to 10.2, 3.56 to 4.88, 9.52 to 16.4, 0.79 to 17.8, 1.03 to 12.0, 0.75 to 3.19, 2.48 to 8.74, 1.93 to 2.54, 12.6 to 15.1, 2.29 to 2.35.
The fingerprint comprises 12 fingerprint peaks, and the relative retention time (RRt, the ratio of the retention time of a single peak to the retention time of the pseudostellaria cyclic peptide B peak) range and the relative peak area of the pseudostellaria cyclic peptide B peak (7) relative to a reference peak are respectively shown in the following table:
wherein, 6: pseudostellarin B; 7: pseudostellaria cyclic peptide B; 8: pseudostellarin C; 9: pseudostellarine E.
The fingerprint comprises 12 fingerprint peaks, wherein the percentage of each peak area to the total peak area is shown in the following table
Wherein, 6: pseudostellarin B; 7: pseudostellaria cyclic peptide B; 8: pseudostellarin C; 9-Pseudomonas callalinin E.
The fingerprint and chemical components of the radix pseudostellariae medicinal material provided by the invention can be applied to the quality control of the radix pseudostellariae medicinal material.
After the fingerprint of the radix pseudostellariae medicinal material is obtained by the method, the sample spectrum is prepared according to the same conditions. The evaluation criteria were: 1. fingerprint comparison: (1) similarity calculation is carried out on the test sample spectrum and the comparison fingerprint spectrum, and the similarity is not lower than 0.9; (2) 12 fingerprint peaks in the test sample map, wherein the peak 7 corresponding to the reference sample is an S peak, and the relative retention time of each fingerprint peak and the S peak is calculated and is within +/-5% of a specified value; (3) comparing the sample spectrum with the fingerprint spectrum, the total area of the non-common peak is not more than 20% of the total peak area. 2. And (3) feature spectrum comparison: the preparation method of the characteristic spectrum is the same as that of the fingerprint spectrum. The test sample characteristic spectrum should have 12 characteristic peaks, the peak 7 corresponding to the reference substance is the S peak, the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within +/-5% of the specified value. Calculating the percentage of each peak area from the peak 1 to the peak 12 to the total peak area, wherein the percentage is within a specified range which is as follows (%): 80 percent.
The invention establishes the determination method for simultaneously determining various components in the radix pseudostellariae, the sample pretreatment is simple, the chromatographic conditions are specific to various compounds, and the identification of 8 compounds is completed.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is an HPLC chromatogram of a sample solvent blank experiment according to example 1 of the present invention.
Fig. 2 is an HPLC chromatogram of the determination result of the measurement time of the sample according to example 1 of the present invention.
FIG. 3 is an HPLC chromatogram of radix Pseudostellariae medicinal material according to example 1 of the present invention, wherein R is a control solution; S1-S3 are sample numbers. Wherein, 6: pseudostellarin B; 7: pseudostellaria cyclic peptide B; 8: pseudostellarin C; 9: pseudostellarine E.
Fig. 4 shows the HPLC profile and the generation of common peak pattern profile R of radix pseudostellariae medicinal material according to example 1 of the present invention, wherein S1-S3: sample number (see table 1); r: consensus peak pattern profile; wherein 1-12 are common peaks; 6: pseudostellarin B; 7: pseudostellaria cyclic peptide B; 8: pseudostellarin C; 9: pseudostellarine E.
Fig. 5 is an HPLC chromatogram of radix pseudostellariae medicinal materials with different qualities detected by using the method of the present invention in example 2, the HPLC chromatogram of a new radix pseudostellariae product is S4, the HPLC chromatogram of an old radix pseudostellariae product is S5, the HPLC chromatogram of a water-extracted radix pseudostellariae residue is S6, the HPLC chromatogram of a water-extracted radix pseudostellariae-containing residue is S7, and the HPLC chromatogram of a radix pseudostellariae-containing radix ophiopogonis is S8.
Fig. 6 is an HPLC chromatogram of different radix pseudostellariae medicinal materials of comparative example 1, wherein an HPLC chromatogram of a new radix pseudostellariae stock is S9, an HPLC chromatogram of a old radix pseudostellariae stock is S10, an HPLC chromatogram of a water-extracted residue of radix pseudostellariae is S11, an HPLC chromatogram of an aqueous-extracted residue-doped radix pseudostellariae is S12, and an HPLC chromatogram of an radix pseudostellariae doped with radix ophiopogonis is S13.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments thereof, which will be better understood from the following examples. However, it should be readily understood by those skilled in the art that the following examples are illustrative only and are not intended to limit the present invention to these specific embodiments. It will be appreciated by those skilled in the art that the present invention encompasses all modifications, alternatives, and equivalents as may be included within the scope of the claims.
Example 1 establishment of Standard fingerprint of radix Pseudostellariae
Instrument, reagent and sample
Agilent Technologies 1260 analytical high performance liquid chromatograph (Agilent Technologies, Inc., USA); KH-500DE digital control ultrasonic cleaner (Kunshan city-Hepiai Co., China); one hundred thousand analytical balances of the XS105 type (Mettler, Switzerland); model AL104 ten thousandth analytical balance (mettler, switzerland).
Comparison products: radix pseudostellariae cyclic peptide B (Heterophyllin B, lot 8110741, purity 99%) purchased from WUDUPUREI scientific and technological development, Inc.;
acetonitrile is chromatographically pure (Merck, Germany), water is Wahaha purified water, and the rest of the reagents are analytically pure. The information of the radix pseudostellariae medicinal material sample is shown in table 1.
TABLE 1 radix Pseudostellariae medicinal material sample information
II, an experimental method:
1. preparation of test solution
Taking a radix pseudostellariae sample medicinal material, and crushing (sieving by a 60-mesh sieve). Weighing 0.5g of powder, adding 10mL of methanol, weighing, performing ultrasonic treatment for 30min, taking out, supplementing weight with methanol, filtering (0.45 μm filter membrane), and taking out the subsequent filtrate.
2. Preparation of control solutions
Accurately weighing 1.00mg of radix Pseudostellariae cyclic peptide B reference substance in a 50mL measuring flask, adding methanol to constant volume, and filtering (0.45 μm filter membrane).
3. Chromatographic conditions
Adopting a YMC-Triart C18 chromatographic column (4.6mm multiplied by 250mm, 5 mu m, Japan YMC company), taking acetonitrile-water as a mixed mobile phase, carrying out gradient elution (table 2), wherein the detection wavelength is 203nm, the column temperature is 25 ℃, the flow rate of the mobile phase is 1.0mL/min, the sample introduction amount is 10 mu L, and the theoretical plate number is not lower than 5000 according to the peak calculation of the pseudostellaria cyclic peptide B.
TABLE 2 gradient elution conditions
4. Methodology investigation
(1) Blank test
Precisely sucking 10 mu L of methanol, injecting the methanol into a high performance liquid chromatograph, and measuring according to chromatographic conditions, wherein the result is shown in figure 1, and the sample solvent has no interference on the measurement result.
(2) Determination of the measurement time
Precisely sucking 10 μ L of the sample solution, injecting into high performance liquid chromatograph, measuring according to chromatographic conditions, and continuously eluting with 90% acetonitrile for 30min after 55 min. As a result, as shown in FIG. 2, no peak was observed after a retention time of 55min, and the retention time was determined to be 55 min.
(3) Precision test
Precisely sucking 10 mu L of the same sample solution, continuously feeding samples for 6 times, determining a fingerprint, taking the radix pseudostellariae cyclic peptide B (peak 7) as a reference peak, calculating the relative retention time (the ratio of each peak to the retention time of the radix pseudostellariae cyclic peptide B) and the relative peak area (the ratio of each peak to the peak area of the radix pseudostellariae cyclic peptide B) of each main peak (figure 3) accounting for more than 2 percent of the total peak area, and obtaining the results shown in Table 3. The RSD of each main peak relative to the retention time is between 0.016 percent and 0.162 percent, the RSD of the relative peak area is between 0.749 percent and 2.746 percent, and the precision of the instrument is good.
TABLE 3 results of the precision test
(4) Stability test
Taking 10 μ L of the same test solution, injecting samples at 0, 6, 12, 24, and 48h respectively, determining fingerprint, taking Ethensenecin B as reference peak, calculating relative retention time and relative peak area of each main peak (shown in figure 3) accounting for more than 2% of total peak area, and the result is shown in Table 4. The RSD of each main peak relative to the retention time is 0.048-0.966%, and the RSD of the relative peak area is 0.893-2.800%, which indicates that the test solution is stable within 48 h.
Table 4 stability test results
(5) Repeatability test
Precisely weighing 5 parts of the same sample, respectively preparing a test solution, measuring a fingerprint, taking the pseudostellaria cyclic peptide B as a reference peak, calculating the relative retention time and the relative peak area of each main peak (figure 3) accounting for more than 2% of the total peak area, and obtaining the results shown in table 5. The RSD of each main peak relative retention time is 0.012-0.773%, and the RSD of each main peak relative peak area is 1.154-2.833%, which are all less than 3%, indicating that the method has good repeatability.
TABLE 5 results of repeated experiments
5. Establishment and analysis of HPLC fingerprint of radix pseudostellariae medicinal material
(1) Establishment of fingerprint
As mentioned above, radix Pseudostellariae medicinal material reference solution and test solution are prepared, measured according to chromatographic conditions, and chromatogram is recorded. As can be seen from fig. 3, the chromatographic peak of pseudostellaria heterophylla cyclic peptide B (peak 7, retention time 24.54min) was well separated and was a common peak, so pseudostellaria heterophylla cyclic peptide B was selected as the reference peak. The fingerprint of the radix pseudostellariae medicinal material is inspected by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), 12 common peaks accounting for more than 2% of the total peak area are totally observed, 4 of the peaks are considered as characteristic peaks and are pseudostellaria cyclic peptide B (peak 6), pseudostellaria cyclic peptide B (peak 7), pseudostellaria cyclic peptide C (peak 8) and pseudostellaria E (peak 9), and the peak 6, the peak 8 and the peak 9 are identified by LC/MS.
(2) Similarity analysis of fingerprint
By adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), three batches of radix pseudostellariae medicinal material HPLC (high performance liquid chromatography) spectra are led into software, and retention time proofreading and similarity calculation are carried out (figure 4, table 6). The comparison shows that the similarity of the radix pseudostellariae samples S1 and S2 is 0.985, the similarity of S3 and S1 and S2 is 0.593 and 0.645, which shows that the quality consistency of S1 and S2 is good, and the quality of S3 and S1 and S2 are greatly different. From the experimental results, the content of the cyclic peptide B (peak 7) in the radix pseudostellariae produced in Guizhou and Jiangsu (S3) is high, while the content of the peak 7 produced in Guizhou (S1) is small, but the content of the peak 6 is high.
TABLE 6 similarity of HPLC fingerprint of radix Pseudostellariae
(3) Analysis of relative retention time and peak area ratio of finger print
For 12 common peaks in the pseudostellaria root medicinal materials, the relative retention time (single peak retention time/reference peak retention time) and the relative peak area (single peak area/reference peak area) of each common peak and the reference peak are respectively determined by taking peak 7 (pseudostellaria cyclic peptide B) as the reference peak (Table 7).
TABLE 7 relative retention time and relative peak area of common peaks of radix Pseudostellariae
Wherein, 6: pseudostellarin B; 7: pseudostellaria cyclic peptide B; 8: pseudostellarin C; 9: pseudostellarine E.
(4) Common fingerprint peak area ratio analysis
The percentage of each peak area in the HPLC profile to the total peak area was calculated for 12 common peaks in the pseudostellaria root drug material, respectively (table 8).
TABLE 8 common fingerprint Peak area ratio analysis
Wherein, 6: pseudostellarin B; 7: pseudostellaria cyclic peptide B; 8: pseudostellarin C; 9: pseudostellarine E.
6. Discussion of results
From the similarity evaluation results of three batches of radix pseudostellariae medicinal materials, in the similarity evaluation result obtained by adopting a traditional Chinese medicine chromatography fingerprint similarity evaluation system (2012 edition), the similarity of radix pseudostellariae samples S1 and S2 is 0.985, and the radix pseudostellariae samples may be produced in Guizhou; the similarity between S3 produced in Jiangsu and S1 and S2 is only 0.593 and 0.645, which indicates that the quality difference is large. From the relative retention time and the relative peak area, the RSD of the relative retention time of 12 common peaks of three batches of radix pseudostellariae medicinal materials is 0.004-0.240%, the relative retention time of each peak is stable, the position of each peak can be calibrated, and the method can be used for evaluating the consistency or stability of the quality of the radix pseudostellariae medicinal materials as long as a radix pseudostellariae cyclic peptide B reference substance exists.
From the relative peak areas of 12 common peaks of three batches of medicinal materials, the RSD of the medicinal materials is over 64 percent, which indicates that the quality difference of the medicinal materials is large. From the ratio of the total peak area of the common peak and the non-common peak of the medicinal materials, the common peak area of the three batches of radix pseudostellariae medicinal materials is 77.1%, 80.8% and 70.3% respectively; among them, the peak areas of 4 characteristic peaks are greatly different, and peak 6(pseudostellarin B) has peak areas of 17.8% and 15.7% in S1 and S2 and 0.79% in S3; peak 7 (pseudostellaria cyclic peptide B) had peak areas of 1.37% and 1.03% in S1 and S2, and 12.0% in S3; the peak area ratio of peak 8 (pseudostellarian C) and peak 9(pseudostellarin E) in S1 and S2 is much larger than that in S3, which reflects that the pseudostellaria root S1, S2 and S3 have great difference, and simultaneously shows that the method can well reflect the quality consistency or difference of the pseudostellaria root medicinal materials.
Example 2 radix Pseudostellariae medicinal materials with different qualities are detected by using the method of the invention
The chromatographic conditions were the same as in example 1 except that the column was changed to Agilent EC-C18(4.6 mm. times.250 mm, 5 μm).
Preparation of control solutions: the same as in example 1.
The test samples are selected from new radix Pseudostellariae, old radix Pseudostellariae, residue obtained by extracting radix Pseudostellariae with water, radix Pseudostellariae mixed with water and residue obtained by extracting radix Pseudostellariae with water, and radix Pseudostellariae mixed with radix Ophiopogonis. Wherein the radix pseudostellariae dregs after the dregs are extracted by the mixed water account for 30 percent of the total mass, and the mixed radix pseudostellariae and the radix pseudostellariae used for extracting the dregs are the same new batch of radix pseudostellariae;
radix Ophiopogonis accounts for 20% of the total weight of radix Pseudostellariae doped with radix Ophiopogonis;
the old pseudostellaria root is purchased and reserved in 2017 in 1 month;
the new radix Pseudostellariae is purchased in 11 months in 2020.
Preparation of a test solution: respectively taking new radix Pseudostellariae, old radix Pseudostellariae, water-extracted residue of radix Pseudostellariae, water-extracted residue-extracted radix Pseudostellariae, radix Pseudostellariae doped with radix Ophiopogonis, and pulverizing (sieving with 60 mesh sieve). Weighing powder 0.5g, adding methanol 10mL, weighing, ultrasonic treating for 30min, taking out, supplementing with methanol, filtering (0.45 μm filter membrane), and collecting filtrate.
The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring to obtain the final product in the same way as example 1.
The experimental results are as follows: referring to fig. 5, the HPLC chromatogram of the new product of radix pseudostellariae is S4, the HPLC chromatogram of the old product of radix pseudostellariae is S5, the HPLC chromatogram of the water-extracted residue of radix pseudostellariae is S6, the HPLC chromatogram of the water-extracted residue of radix pseudostellariae is S7, and the HPLC chromatogram of the radix pseudostellariae doped with radix ophiopogonis is S8.
The experimental results are as follows: as can be seen from fig. 5, 12 characteristic peaks are represented in both the new and old radix pseudostellariae products, but the area of the 11 characteristic peak in the new radix pseudostellariae product is significantly higher than that of the old radix pseudostellariae product, no 2, no 3, no 4, or no 6 characteristic peaks are missing in the radix pseudostellariae dregs after water extraction, the ratio of the area of the 2, no 3, no 4, or no 6 characteristic peak of the radix pseudostellariae after the dregs are extracted with water to the area of the 7 characteristic peak is not consistent with the ratio of the area of the above characteristic peak of the new radix pseudostellariae product, and unknown chromatographic peaks are detected in the 2 to 7 peaks of the radix pseudostellariae doped with radix ophiopogonis.
Therefore, the method has a good separation effect, the radix pseudostellariae fingerprint provided by the invention has a stable baseline, a large number of chromatographic peaks, and compounds with different polarity sections are well reflected. Moreover, according to the method and the fingerprint provided by the invention, a good identification effect is realized on different qualities of radix pseudostellariae and adulterated radix pseudostellariae medicinal materials, and the characterization of various complex components in the radix pseudostellariae medicinal materials is easier to realize.
Comparative example 1 radix pseudostellariae medicinal materials with different qualities are detected by using the method in the prior art
Referring to the method reported by Chizhibiao et al in the research on HPLC fingerprint of radix pseudostellariae, the specific method is as follows:
chromatographic conditions are as follows: an Agilent EC-C18 chromatographic column (4.6mm × 250mm, 5 μm) with a column temperature of 25 ℃, acetonitrile as phase A and water as phase B was used for gradient elution with a flow rate of 1ml/min and a detection wavelength of 203 nm.
The elution gradient is shown in table 9:
TABLE 9 elution gradient
The test samples are selected from new radix Pseudostellariae, old radix Pseudostellariae, residue obtained by extracting radix Pseudostellariae with water, radix Pseudostellariae mixed with water and residue obtained by extracting radix Pseudostellariae with water, and radix Pseudostellariae mixed with radix Ophiopogonis. Wherein the radix pseudostellariae dregs after the dregs are extracted by the mixed water account for 30 percent of the total mass, and the mixed radix pseudostellariae and the radix pseudostellariae used for extracting the dregs are the same new batch of radix pseudostellariae;
radix Ophiopogonis accounts for 20% of the total weight of radix Pseudostellariae doped with radix Ophiopogonis;
the old pseudostellaria root is purchased and reserved in 2017 in 1 month;
the new radix Pseudostellariae is purchased in 11 months in 2020.
Preparation of a test solution:
respectively taking new radix pseudostellariae, old radix pseudostellariae, water-extracted radix pseudostellariae dregs and radix pseudostellariae doped with radix ophiopogonis, adding 30mL of ethyl acetate, carrying out ultrasonic extraction for 40min at 300W, cooling, filtering, adding 30mL of ethyl acetate into filter residues, washing for 3 times, combining filtrate and washing liquor, steaming at 60 ℃ till the mixture is nearly dry, adding 0.3g of polyamide (60-80 meshes) and stirring uniformly, adding the mixture onto 1g of polyamide column (1.5cm of filtrate and washing liquor), eluting with 50mL of water, 50mL of 30% ethanol and 50mL of 80% ethanol respectively, collecting eluent of 80% ethanol, drying by distillation, dissolving with methanol, and putting the mixture into a 2mL measuring flask. Passing through 0.45m microporous filter membrane before use as test sample.
The determination method comprises the following steps: precisely sucking 10 μ l of the sample solution, injecting into liquid chromatograph, and measuring.
The experimental results are as follows: referring to fig. 6, the HPLC chromatogram of the new product of radix pseudostellariae is S9, the HPLC chromatogram of the old product of radix pseudostellariae is S10, the HPLC chromatogram of the water-extracted residue of radix pseudostellariae is S11, the HPLC chromatogram of the water-extracted residue of radix pseudostellariae is S12, and the HPLC chromatogram of the radix pseudostellariae doped with radix ophiopogonis is S13.
Therefore, the column chromatography method is adopted in the pretreatment, so that the high selectivity of the components of the radix pseudostellariae medicinal materials exists, the maps with higher similarity are obtained for different qualities and adulterated radix pseudostellariae, and the inherent quality of the radix pseudostellariae medicinal materials cannot be better distinguished. Compared with the scheme, the embodiment 1 has the advantages that the sample treatment is simple, the chromatographic peak characterization is good, the radix pseudostellariae chromatograms with different qualities have obvious differences, and the adulteration of the radix pseudostellariae can be well distinguished.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (9)
1. A fingerprint spectrum establishing method of radix pseudostellariae medicinal materials adopts high performance liquid chromatography, and the conditions of the high performance liquid chromatography comprise: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is used as a mobile phase A, water is used as a mobile phase B to form a mixed mobile phase, and the volume ratio of the mobile phase A to the mobile phase B in the mixed mobile phase within 0-55 min is from 5: 95 to 90: gradient elution with a gradient of 10; the detection wavelength was 203 nm.
2. The fingerprint spectrum establishing method according to claim 1, wherein said gradient is as follows:
3. the fingerprint establishment method according to claim 1 or 2, wherein the flow rate of said mixed mobile phase is 1 mL/min.
4. The fingerprint establishment method according to any one of claims 1 to 3, wherein the conditions of said high performance liquid chromatography further comprise: the column temperature is 20-40 ℃; the sample injection amount is 10 mu L; preferably, the column temperature is 25 ℃.
5. The fingerprint establishment method according to any one of claims 1 to 4, comprising the steps of:
(1) preparation of control solutions: taking a radix pseudostellariae cyclic peptide B reference substance, and adding methanol to prepare a reference substance solution;
(2) preparing a test solution: taking radix pseudostellariae medicinal material, adding methanol, carrying out ultrasonic treatment, filtering, and taking subsequent filtrate to obtain a test solution;
(3) the determination method comprises the following steps: injecting the reference substance solution prepared in the step (1) and the test solution prepared in the step (2) into a high performance liquid chromatograph, and obtaining a high performance liquid chromatogram according to the conditions of the high performance liquid chromatography;
more preferably, the fingerprint establishing method comprises the following steps:
(1) preparation of control solutions: taking a radix pseudostellariae cyclic peptide B reference substance, and adding methanol to prepare a reference substance solution containing 0.02mg of radix pseudostellariae cyclic peptide B per 1 mL;
(2) preparing a test solution: taking 0.5g of radix pseudostellariae, adding 10mL of methanol, carrying out ultrasonic treatment for 10-60 minutes, such as 30 minutes, cooling, supplementing the lost weight with methanol, shaking up, filtering, and taking the subsequent filtrate to obtain a test solution;
(3) the determination method comprises the following steps: injecting the reference substance solution prepared in the step (1) and the test solution prepared in the step (2) into a high performance liquid chromatograph, and obtaining a high performance liquid chromatogram according to the conditions of the high performance liquid chromatography;
preferably, the method further comprises the steps of obtaining high performance liquid chromatography maps of a plurality of radix pseudostellariae medicinal material samples according to the conditions of the high performance liquid chromatography, checking and matching retention time of common chromatographic peaks in the high performance liquid chromatography maps by selecting reference peaks in the high performance liquid chromatography maps, calculating similarity, and generating common peak mode maps.
6. A method for detecting active ingredients of a radix pseudostellariae medicinal material comprises the following steps:
1) obtaining a high performance liquid chromatography fingerprint by using the radix pseudostellariae medicinal material fingerprint establishing method according to any one of claims 1 to 5, obtaining a high performance liquid chromatography of a sample to be detected of the radix pseudostellariae medicinal material according to the conditions of the high performance liquid chromatography of any one of claims 1 to 5, and comparing the high performance liquid chromatography and the high performance liquid chromatography;
2) and identifying the components of the radix pseudostellariae medicinal material sample to be detected by adopting a mass spectrometry method.
7. The radix pseudostellariae medicinal material fingerprint established by the radix pseudostellariae medicinal material fingerprint establishing method according to any one of claims 1 to 5.
8. The radix pseudostellariae medicinal material fingerprint spectrum according to claim 7, wherein the radix pseudostellariae medicinal material fingerprint spectrum at least comprises 12 common characteristic peaks; the relative retention time ranges of the pseudostellaria heterophylla cyclic peptide B peak relative to the reference peak are respectively as follows: 0.505, 0.675-0.678, 0.709, 0.739, 0.789-0.790, 0.941, 1.000, 1.067, 1.217-1.221, 1.956-1.957, 2.030, 2.146-2.147;
preferably, wherein peak No. 5: pseudostellarin a, peak No. 6: pseudostellarin B, peak No. 7: pseudostellaria cyclic peptide B, peak No. 8: pseudostellarin C, peak No. 9: pseudostellarin D, peak No. 10: pseudostellaria cyclic peptide a, peak 11: pseudostellaring, peak No. 12: pseudostellarin E;
preferably, the percentage ranges of each peak area of the 12 common characteristic peaks in the total peak area are respectively as follows: 1.01 to 3.70, 3.72 to 9.03, 4.37 to 10.2, 3.56 to 4.88, 9.52 to 16.4, 0.79 to 17.8, 1.03 to 12.0, 0.75 to 3.19, 2.48 to 8.74, 1.93 to 2.54, 12.6 to 15.1, 2.29 to 2.35.
9. Use of the fingerprint of radix pseudostellariae as claimed in claim 7 or 8 in quality control of radix pseudostellariae.
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| CN118501299B (en) * | 2024-07-18 | 2024-10-11 | 江中药业股份有限公司 | Evaluation method of pseudostellaria root cyclic peptide B content based on electronic nose |
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